Based on the iCE280 technology from Convergent Bioscience, the iCE3 performs free solution isoelectric focusing in a capillary column and detects focused protein zones using a whole column UV detector. This unique technology combines the resolution of traditional gel IEF with the advantages of quantitation and automation found in column based separations while eliminating the need for a mobilization step.
How does it work?
A sample is prepared containing the protein of interest, carrier ampholytes and pI markers. The sample is loaded into a capillary cartridge and the electrolytic tanks at each end of the capillary are filled with acid and base. Voltage is applied and the analytes are focused at their pI. A CCD camera takes a UV light absorption image of the entire capillary column every 30 seconds allowing real time monitoring of the the focusing step. The resulting separation pattern is captured and analyzed.
Fundamentals of IEF
iCE3 utilizes isoelectric focusing or IEF. IEF is a mode of electrophoresis for amphoteric molecules, especially peptides and proteins. It separates molecules purely based on their pI values, in contrast to other electrophoresis techniques which typically utilize some form of size-based separation. IEF is also a concentrating technique since molecules having the same pIs are focused into narrow zones in the pH gradient. At the end of the separation, all these zones are stationary. IEF is also an equilibrium separation technique, and provides the highest resolution of all charge-based separation techniques for proteins.
Traditional Slab Gel IEF
Traditional IEF is performed in polyacrylamide slab gels. First, a pH gradient is created on the slab gel by commercial carrier ampholytes under a separation voltage. Then, protein samples are loaded into the gel to start the separation. Although slab gel IEF has high resolution for protein separation, it tends to be slow, labor intensive, and not quantitative. It was recognized among scientists for a long time that if high-resolution IEF could be performed in a column format, significant advantages over slab gel IEF in terms of automation, separation speed and quantitation could be realized. This was why exploration of cIEF began in 1985.
Conventional capillary IEF
Despite the appeal of cIEF since it was proposed in 1985, widespread acceptance as a replacement for slab gel IEF did not occur. The main factor for the slow acceptance of cIEF was the difficulty in performing cIEF using commercial, general-purpose capillary electrophoresis (CE) instruments (now referred to as conventional cIEF). These commercial CE instruments are equipped with a single point, on-column detector. In conventional cIEF, after the isoelectric focusing process, a mobilization process is necessary to move all the focused protein zones past the detection point of the on-column detector in order to detect these zones. The mobilization process introduces many problems such as poor resolution, poor reproducibility and long sample analysis times (less than 2 samples/hour). In conventional cIEF, the dynamic process of IEF within the separation columnis not monitored, which makes it difficult to optimize focusing time, one of the most important parameters in cIEF. It is also impossible to find problems in the IEF process within the separation column, such as sample aggregation and precipitation.
ProteinSimple (formerly Convergent Bioscience) was the first to commercialize the whole-column detection cIEF technique in the iCE IEF Analyzer instrument. The iCE instrument revolutionized cIEF technology.
In performing cIEF on the iCE instrument, protein samples are first premixed with carrier ampholytes, additives and pI markers. The mixture is injected to fill the entire capillary column. A separation voltage is applied to the anolyte and catholyte tanks. Under the voltage, a pH gradient is created within the column. Proteins are separated and focused along the capillary column. The whole-column detector monitors the IEF process in an on-line fashion within the separation column, and the focusing time can be optimized in a single sample run. At the end of the focusing process, all the focused protein zones within the column are recorded by the detector without disturbing the separation resolution. Finally, the column is washed and ready for the next sample injection. Any sample precipitation and aggregation during focusing can be observed. Different additives are easily selected to improve reproducibility when issues are identified. The biggest advantage of whole column detection cIEF that users have found is its fast and easy method development because of the ability to monitor the IEF process within the separation column.